The sarcoplasmic reticulum (SR) is the major Ca2+-sequestering membrane system responsible for cardiac relaxation and the initiation of contraction. The status of phosphorylation of a SR protein named phospholamban (PLN) determines the activity of the SR-Ca2+-pump and the rate of Ca2+-transport from the cytosol into the SR. Thus, PLN is a fundamental determinant of intracellular Ca2+ handling, cardiac contractility and relaxation. Under the auspice of the FIRCA grant (FIRCA project 1 R03 TW06294-01), we described for the first time a cascade of events that revealed the importance of phosphorylations evoked by the Ca2+-calmodulin-dependent protein kinase (CaMKII), specifically the phosphorylation of Thr17 of PLN, in intracellular Ca2+, contractile and relaxation recoveries, after a short period of ischemia (reversible ischemia/reperfusion (I/R) injury or stunned heart). Reperfusion after a long ischemic period leads to cardiac damage and cell death by necrosis and apoptosis (irreversible I/R injury). Identification of the mechanisms responsible for this irreversible cardiac injury is of great importance for the search of novel strategies for cardioprotection, given that myocardial infarction is one of the leading causes of death in adults of the western countries. The present project proposes the study of the role of the phosphorylation of SR proteins responsible for Ca2+ handling, particularly those dependent on CaMKII activation, as well as the mechanisms and signaling cascades involved in these phosphorylations, in the irreversible I/R injury. To that purpose, transgenic models with specific mutation of the CaMKII-site of PLN, specific inhibition of the SR CaMKII and non-transgenic animals with chronic inhibition of CaMKII, will be used in combination with proteomic techniques and the assessment of contractility and relaxation, infarct size, apoptosis, CaMKII activity and immunodetection of site-specific phosphorylation of SR proteins. This experimental strategy will allow us to define the role of the CaMKII-dependent cascades involved in irreversible I/R injury. The results may form the basis for future preventive and therapeutic approaches at the level of the CaMKII and/or the proteins involved, which may be beneficial to the irreversible I/R injury This research will be done primarily in the Cardiovascular Research Center, La Plata, Argentina, in collaboration with Dr. Evangelia Kranias, as an extension of the parent grant HL26057 (Merit Award). [unreadable] [unreadable] [unreadable]